Petersen Shay, K., Wang, Z., Xing, P.X., McKenzie, I.F. and Magnuson, N.S.
Notes: Through immunoprecipitation studies it was found that levels of the proto-oncogenic serine/threonine kinase Pim-1 are regulated by ubiquitination. The kinase was found to associate with the chaperone proteins Hsp70 and Hsp90. Hsp70 associated with the ubiquitinated form of Pim-1, and Hsp90 protected Pim-1 from degradation. To study the activity of Pim-1 in these associations with chaperone proteins, recombinant Pim-1 protein was mixed with a five-fold excess of recombinant Hsp70 or Hsp90, or left unbound in the presence of a binding buffer [10 mmol/L HEPES (pH 7.4), 100 mmol/L KCl, 5 mmol/L DTT, 20 mmol/L Na2MoO4, 50 mmol/L ATP]. These complexes were then immunoprecipitated with a Pim-1 monoclonal antibody and washed four times in kinase buffer [25 mmol/L HEPES, 10 mmol/L MgCl2, 0.5 μg/mL DTT]. Finally, 0.1μmol/L ATP and 400 μmol/L of peptide substrate were added and the reaction incubated for 5 minutes at 30° C. The Kinase-Glo® Luminescent Kinase Assay was used to determine the activity of Pim-1 in its various bound states. It was found that all binding configurations of the kinase were active, underscoring the importance of efficient degradation of the kinase as a means for regulating its activity. (3264)